Abstract

The ability of cancer cells to degrade the extracellular matrix and invade interstitial tissues contributes to their metastatic potential. We recently showed that overexpression of sorting nexin 9 (SNX9) leads to increased cell invasion and metastasis in animal models, which correlates with increased SNX9 protein expression in metastases from human mammary cancers. Here, we report that SNX9 expression is reduced relative to neighboring normal tissues in primary breast tumors, and progressively reduced in more aggressive stages of non-small-cell lung cancers. We show that SNX9 is localized at invadopodia where it directly binds the invadopodia marker TKS5 and negatively regulates invadopodia formation and function. SNX9 depletion increases invadopodia number and the local recruitment of MT1-MMP by decreasing its internalization. Together, these effects result in increased localized matrix degradation. We further identify SNX9 as a Src kinase substrate and show that this phosphorylation is important for SNX9 activity in regulating cell invasion, but is dispensable for its function in regulating invadopodia. The diversified changes associated with SNX9 expression in cancer highlight its importance as a central regulator of cancer cell behavior.

SNX9 negatively regulates invadopodia formation. (A) 231-CTR or 231-siSNX9 cells were plated on gelatin and stained for cortactin (green) and F-actin (phalloidin, in red) to visualize invadopodia. Insets represent enlargement of yellow boxes. Scale bars: 20 μm, insets 4 μm. (B,C) Quantification of percentage of cells expressing invadopodia (B) or of the relative number of invadopodia (C) in conditions used in A. ∼200 cells were counted for each condition, n=3; *P≤0.05, ****P<0.0001. Results are presented as mean±s.e.m. Statistical significance was evaluated using one-tailed Mann–Whitney test. (D) 231-CTR or 231-siSNX9 cells plated on a filter containing 1-μm-diameter pores. Images represent a z-stack of confocal sections of invadopodia extending through the filter. F-actin is visualized in red and cortactin in green. Two independent siRNAs against SNX9 were used. (E) Western blot analysis of SNX9 expression under conditions used in D showing knockdown of SNX9 expression in MDA-MB-231 cells transfected with SNX9 siRNA #1 and #2. GAPDH was used as loading control.

SNX9 is phosphorylated by Src. (A) NIH-3T3 or NIH-Src were treated with Src inhibitor SU6656 or DMSO (control). Cells were then stained for F-actin and for endogenous SNX9. Scale bars: 10 μm. (B) Western blot comparing exogenous SNX9 phosphorylation in NIH-Src cells transiently expressing GFP–SNX9, mock-treated or treated with SU6656. Tyrosine phosphorylation was evaluated on immunoprecipitated GFP–SNX9 using an anti-phospho-tyrosine antibody. GAPDH was used as loading control. (C) Schematic presentation of SNX9 protein domains. Tyrosines, identified by mass spectrometry (see Fig. S4B), phosphorylated by Src are marked in red. (D) HA–Src and V5–WT-SNX9 or the indicated mutants were transiently co-expressed in HEK293 cells. V5–SNX9 was then immunoprecipitated and tyrosine phosphorylation was assessed using an anti-phospho-tyrosine antibody. 5YF represents the quintuple mutant, where all phosphorylated tyrosines were mutated (Y177F, Y239F, Y269F, Y294F and Y561F).